RNA polymerase of vesicular stomatitis virus specifically associates with translation elongation factor-1 alphabetagamma for its activity.
An RNA-dependent RNA polymerase is packaged within the virions of purified vesicular stomatitis virus, a nonsegmented negative-strand RNA virus, which carries out transcription of the genome RNA into mRNAs both in vitro and in vivo. The RNA polymerase is composed of two virally encoded polypeptides: a large protein L (240 kDa) and a phosphoprotein P (29 kDa). Recently, we obtained biologically active L protein from insect cells following infection by a recombinant baculovirus expressing L gene. During purification of the L protein from Sf21 cells, we obtained in addition to an active L fraction an inactive fraction that required uninfected insect cell extract to restore its activity. The cellular factors have now been purified, characterized, and shown to be beta and gamma subunits of the protein synthesis elongation factor EF-1. We also demonstrate that the alpha subunit of EF-1 remains tightly bound to the L protein in the inactive fraction and betagamma subunits associate with the L(alpha) complex. Further purification of L(alpha) from the inactive fraction revealed that the complex is partially active and is significantly stimulated by the addition of betagamma subunits purified from Sf21 cells. A putative inhibitor(s) appears to co-elute in the inactive fraction that blocked the L(alpha) activity. The purified virions also package all three subunits of EF-1. These findings have a striking similarity with Qbeta RNA phage, which also associates with the bacterial homologue of EF-1 for its replicase function, implicating a possible evolutionary relationship between these host proteins and the RNA-dependent RNA polymerase of RNA viruses.
Das
T
T
Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, NC20, Cleveland, OH 44195, USA.
Mathur
M
M
Gupta
A K
AK
Janssen
G M
GM
Banerjee
A K
AK
D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000201:enzymology*; D014764:Viral Proteins; D014771:Virion / Q000737:chemistry
D046911:Macromolecular Substances; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins; D014764:Viral Proteins; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase